BIO SCI M114L Chapter 1: WEEK 01- LAB NOTES Cytochrome P450, Bacterial Protein Expression System, Plasmid Miniprep, Restriction Enzymes, Cell Free Extract.pdf
7 May 2015
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Week 01- cytochrome p450, bacterial protein expression system, plasmid miniprep, restriction enzymes, cell free extract. Present of heme prosthetic group containing a central iron atom: iron is bound to 4 nitrogen atoms & a cysteine. Heme group is important for enzyme function. Functions as hydroxylases (monooxygenases) catalyzes the replacement of a substrates"s h w/ oh. Consist of a single protein and 2 distinct domains: contains heme group & fad, fmn. Fad domain is also responsible for nadph binding. P450 protein: highly conserved, expressed in bacteria humans, 100"s diff. enzymes, plays a role of many diff. functions. Substrate hydroxylation: involves transfer of e-s one o atom in o is reduced to a hydroxyl group, second o is reduced to water, e-s are req. Are passed to enzyme"s active site by nadh/nadph. In lab: will get 2 tubes of . coli. 1 expressing p450 gene pet3 vector w/ pt7bm3. 1 expressing an empty vector (no p450 gene) pet3 vector.
